Amyloid beta-peptide (Abeta) is a major component of amyloid plaques in Alzheimer's disease. Abeta is formed through intracellular proteolytic processing of a membrane-anchored glycoprotein termed beta-amyloid precursor protein (APP). Cells can produce alternate forms of Abeta (e.g., Abeta40 and Abeta42), and Abeta42 has the greatest tendency to form insoluble deposits. Our general hypothesis is that the relative amounts of Abeta42 and Abeta40 generated and released by the cell are determined, at least in part, at the level of protein trafficking. Our long range goal is to define the specific transport events that are critical for (1) the amyloidogenic processing of APP C-terminal fragments by intracellular protease(s) termed gamma-secretase, and (2) the subsequent release of different forms of Abeta from the cell. To pursue this goal, molecular and viral transfection strategies will be used to express dominant-negative Rab mutants in cultured cells. Since different Rab GTPases function as mediators of vesicular transport between specific donor and acceptor compartments in the exocytic and endocytic pathways, this strategy will allow us to selectively disrupt discrete trafficking steps that may underlie the delivery of precursor peptides to organelles containing gamma-secretase activity and the delivery of the final Abeta products to the extracellular environment. To facilitate the identification of steps that may vary in neurons versus non-differentiated cells, studies will be carried out in both NT2N neurons and human embryonal kidney cells (HEK293). By expressing various Rab mutants with altered forms of APP that harbor mutations found in familial Alzheimer's disease, it should be possible to identify specific trafficking steps that have particular relevance for the increased production of Abeta from these altered precursors. These studies will provide new information about the subcellular compartmentalization of the gamma-secretase activities that give rise to different forms of Abeta, and help define the routes whereby these products are released from the cell. This information may facilitate the development of therapeutic strategies targeted at the relevant proteases, particularly the gamma-secretase responsible for the production of the pathogenic Abeta42 isoform.